The goal of this study is to identify proteins involved in maintaining the regulated activity and localization of the dopamine (DA) transporter (DAT) at presynaptic nerve terminals. DA is a modulator of both excitatory and inhibitory neurotransmission in the CNS and is critically involved in the pathophysiology of several neuropsychiatric diseases including drug addiction and schizophrenia. DAT is a major determinant of synaptic DA inactivation, an important target for psychostimulant drugs, and a gateway for several neurotoxins. The proper expression and activity of DAT is therefore essential in maintaining normal DA homeostasis in the brain. DATs are subject to acute regulation and are known to form complexes with accessory proteins to control localization and activity, but the mechanisms mediating these processes m vivo are largely unknown. Further, though numerous studies have identified proteins that associate with DAT, no reports to date have demonstrated a functional relevance for these interactions. The intricacy of the mammalian CNS has encouraged our laboratory to pursue such questions in Caenorhabditis elegans. The high degree of conservation between invertebrate and vertebrate neurons at the molecular level suggests that analysis of the C. elegans DAT in living DA neurons could lead to novel insights of general relevance for DAT regulatory processes and DAT-supported drug addiction in man. In this proposal, I present two strategies for the identification of DAT-interacting proteins: first, I will employ RNA-based interference (RNAi) to disrupt the expression of orthologs that have been identified as putative DAT regulators; second, using a novel motor phenotype that I have identified and characterized, I will implement an unbiased, forward genetic screen. C. elegans offers considerable advantages over more complex vertebrate model systems, which make the nematode a valuable tool for identifying DAT-interacting proteins while simultaneously examining their functional relevance. The studies included in this proposal will serve as an excellent vehicle with which to advance my training in model systems genetics and DA neurotransmission and are an ideal avenue for my transition into research independence. Public: Alterations in dopaminergic (DA) neurotransmission are centrally involved in the pathophysiology of several neuropsychiatric diseases, including Parkinson's Disease, Schizophrenia, and drug addiction. Uptake via the presynaptic DA transporter (DAT) is the primary mechanism by which DA signaling at the synapse is terminated, and a thorough understanding of the processes that regulate DAT activity are critical in interpreting the etiology and progression of DA-related diseases. This research will define mechanisms that regulate DAT activity and will aid in the development of treatments for these diseases. ? ? ?
